Journal: Frontiers in Immunology

Article Title: Piezo1 protects against inflammatory bone loss via a unique Ca 2+ -independent mechanism in osteoclasts

doi: 10.3389/fimmu.2025.1661538

Figure Lengend Snippet: Piezo 1 activation by Yoda1 strongly suppressed Akt phosphorylation. (A) Phospho Explorer Antibody Array was performed to determine Yoda1-mediated signaling pathway in pre-OCs. KEGG and IPA were used for bioinformatics analysis. (B) Pre-OCs were stimulated with RANKL (10 ng/ml) in the presence or absence of Yoda1 (5 uM) for 30 min to monitor protein phosphorylation, including Akt, GSK-3b, PI3K, p38 MAPK , ERK, JNK and NF-kB. siRNA-transfected OCs were cultured with shear flow, and samples were collected to monitor Akt phosphorylation by Western blotting. Densitometric analysis was conducted using ImageJ software (Version 1.50). (C) Pre-OCs from Piezo1 flox/flox mice or Piezo1 LysMΔ mice were stimulated with Yoda1 for the indicated time courses, and Akt phosphorylation was assessed by Western blotting. (D) Pre-OCs treated with siRNA for Piezo1 or negative control were stimulated with shear stress at 20 dyn/cm 2 , and then Western blotting was preformed to determine Akt phosphorylation. (E) Pre-OCs were stimulated with Yoda1 for 30min, and cell lysates were subjected to Co-IP to analyze the interaction between RANK and TRAF6. Representative band images are shown from three independent experiments. Data represent the mean ± SD of three independent experiments. *p < 0.05 **p < 0.01 ***p < 0.001 ****p < 0.0001.

Article Snippet: The detection of specific proteins in pre-OCs was assessed using anti-phospho-Akt rabbit mAb (193H12, 1:1000; Cell Signaling Technology, Danvers, MA), anti-Akt rabbit mAb (C67E7, 1:1000; Cell Signaling Technology), anti-phospho-p38 MAPKs rabbit mAb (3D7, 1:1000; Cell Signaling Technology), anti-p38 MAPKs rabbit mAb (D13E1, 1:1000; Cell Signaling Technology), anti-phospho-ERK rabbit mAb (D13.14.4E, 1:2000; Cell Signaling Technology), anti-ERK rabbit mAb (137F5, 1:2000; Cell Signaling Technology), anti-phospho-JNK rabbit mAb (81E11 , 1:1000; Cell Signaling Technology), anti-JNK rabbit mAb (9258, 1:1000; Cell Signaling Technology), anti-IkBa rabbit mAb (L35A5, 1:1000; Cell Signaling Technology), anti-phospho-PP2A mouse mAb (F-8, 1:1000; Santa Cruz Biotechnology, Dallas, TX), anti-PP2A mouse mAb (6F9, 1:1000; Santa Cruz Biotechnology) or an anti-GAPDH rabbit mAb (14C10, Cell Signaling Technology).

Techniques: Activation Assay, Phospho-proteomics, Ab Array, Transfection, Cell Culture, Shear, Western Blot, Software, Negative Control, Co-Immunoprecipitation Assay